EP1448766A2 - Collections de cellules eucaryotes co-isogeniques - Google Patents

Collections de cellules eucaryotes co-isogeniques

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Publication number
EP1448766A2
EP1448766A2 EP02773683A EP02773683A EP1448766A2 EP 1448766 A2 EP1448766 A2 EP 1448766A2 EP 02773683 A EP02773683 A EP 02773683A EP 02773683 A EP02773683 A EP 02773683A EP 1448766 A2 EP1448766 A2 EP 1448766A2
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EP
European Patent Office
Prior art keywords
cells
cell
coisogenic
target locus
genotypically distinct
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Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
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EP02773683A
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German (de)
English (en)
Inventor
Eric B. Kmiec
Michael C. Rice
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University of Delaware
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University of Delaware
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes

Definitions

  • the present invention is in the field of molecular biology, and relates to coisogenic eukaryotic cell collections and methods of use therefor. More specifically, the invention relates to collections of eukaryotic cells that have been engineered to differ from l o one another by as few as one encoded amino acid at a defined target locus, particularly, but not exclusively, target loci that encode proteins that affect responsiveness to therapeutic agents, and to pharmacogenomic methods based thereupon.
  • the newly-emerging field of pharmacogenomics is premised on the notion that statistical correlations of genotypic variations that occur naturally within a population (allelic variation) with their respective phenotypes can be used to predict an individual 5 patient's responsiveness to therapy based upon knowledge of the patient's genotype; the ultimate goal is to stratify patient populations into genetic cohorts for which therapy can be separately tailored. See, e.g., Adam et a/., "Pharmacogenomics to predict drug response," Pharmacogenomics 1(1):5-14 (2000); Judson et al., "The predictive power of haplotypes in clinical response," Pharmacogenomics 1 (1):15-26 (2000).
  • cytochrome 450 enzyme encoded by CYP2D6 is known to metabolize as many as 20% of commonly prescribed drugs.
  • the gene is highly polymorphic in the population; certain alleles result in the poor metabolizer phenotype, characterized by a decreased ability to metabolize the enzyme's substrates.
  • In vitro assays have been developed to assess the drug sensitivity of individual cells. For example, U.S. Patent Nos.
  • Genetic modifications that have typically been contemplated for eukaryotic cells used in screening assays include targeted deletion or disruption of genes, dominant negative suppression of gene expression, and change in gene copy number. See, e.g., U.S. Patent Nos. 5,569,588, 5,777,888, 6,165,709, 6,046,002.
  • yeast notably Saccharomyces cerevisiae
  • the chosen modification leaves heterologous nucleic acids at or near the target locus, a legacy of virally-mediated modification events. See, e.g., U.S. Patent No. 6,207,371.
  • the present invention satisfies these and other objects in the art by providing, in a first aspect a collection of cultured cells, comprising at least 5, 10, or at least 25 genotypically distinct cells, wherein each of the genotypically distinct cells is coisogenic with respect to the others in the collection at a common target locus.
  • the genotypically distinct cells of the collection are separately assayable.
  • two genotypically distinct cells are “coisogenic” with respect to one another if derived from a common ancestor cell and engineered to differ from one another in genomic sequence at a predetermined target locus.
  • the genomic sequence differences at the target locus must be sufficient to alter the amino acid sequence encoded at the target locus by at least one amino acid.
  • the term "coisogenic" permits of changes as between the genomes of the genotypically distinct cells additional to the changes at the target locus.
  • the coisogenic cells of the collection are
  • the cells are alternatively, or additionally, legacy-free, that is, lacking in heterologous genetic elements within 10 kilobases of any codon of the target locus.
  • the coisogenic cells can be from any eukaryote; although usefully mammalian, especially human, the cells can also be of yeast or plant origin. 5
  • the genotypically distinct cells of the collection collectively include each of the 20 natural amino acids at a single residue encoded at the target locus.
  • the genotypically distinct cells collectively include a predetermined amino acid at each residue encoded after the initiator methionine at the target locus.
  • the genotypically distinct cells o collectively include at least one, and on occasion a plurality, of naturally occurring allele of the target locus.
  • the cells of the collection can further comprise a common selectable marker at a genomic locus different from said target locus, and/or a marker unique to said genotypically distinct cell, the unique marker being at a locus different from the target locus.
  • the target locus can be any locus of interest, and in particularly useful embodiments, is selected from the group of loci affecting drug resistance (sensitivity) or drug metabolism consisting of: CYP1A2, CYP2C17, CYP2D6, CYP2E, CYP3A4, CYP4A11 , CYP1B1 , CYP1A1 , CYP2A6, CYP2A13, CYP2B6, CYP2C8, CYP2C9, CYP11A, CYP2C19, CYP2F1, CYP2J2, CYP3A5, CYP3A7, CYP4B1, CYP4F2, CYP4F3, CYP6D1 , CYP
  • the invention provides the coisogenic cell collection in the form of a kit.
  • the kit comprises at least five genotypically distinct cells, the cells contained within separate, structurally discrete, fluidly noncommunicating containers, wherein each of the genotypically distinct cells is coisogenic with respect the others at a o target locus common thereamong; the structurally discrete containers are commonly packaged.
  • the kit further comprises a computer readable medium, recorded upon which is a dataset (typically, a relational database) that describes the target locus genotype of each of said genotypically distinct cells.
  • the invention provides a method of making a coisogenic 5 cell collection.
  • the method comprises collecting at least 5 genotypically distinct cells, each of the genotypically distinct cells being coisogenic with respect to the others at a target locus common thereamong, into a collection in which each of the genotypically distinct cells can be separately assayed.
  • the coisogenic cells will first be prepared, and the method will thus further comprise the antecedent step of engineering, into at least four of five cultured cells, the cells having derived from a common eukaryotic ancestor cell, a genomic sequence alteration at a target locus common thereamong.
  • the sequence alterations should be sufficient to cause at least five distinct protein 5 sequences collectively to be encoded by the cells at the target locus.
  • the engineering is effected by introducing a targeting oligonucleotide into each of said at least four cultured cells.
  • the targeting oligonucleotide effects site-specific change to the cellular genomic DNA.
  • a targeting oligonucleotide is first used to effect a change in a genomic o recombination-competent substrate, such as an artificial chromosome, and the recombination-competent substrate then introduced into each of the four cultured cells.
  • the invention provides a kit useful for creating the coisogenic cell collections of the present invention.
  • the kit comprises at least four targeting oligonucleotides of distinct sequence; and a eukaryotic cell.
  • the targeting oligonucleotides 5 are sufficient to effect four different sequence changes, each sequence change sufficient to alter the protein sequence, at the target genomic locus.
  • the coisogenic cell collections of the present invention can be used for multiplex, including high throughput multiplex screening for mutations that affect a cellular phenotype in vitro.
  • the invention provides a method of identifying genotypes of a target locus that alter a cellular phenotype, comprising a first step of assaying each genotypically distinct cell of a coisogenic cell collection for a common phenotypic characteristic.
  • the genotypically distinct cells are coisogenic at the target locus, preferably exceptionally or perfectly coisogenic, and/or legacy-free.
  • the method calls for identifying from the assay results at least one cell having an altered 5 phenotypic characteristic; and correlating, for the cell or cells with altered phenotypic characteristic, the results of said phenotypic assay with the cell's target locus genotype.
  • Such correlation of phenotypic assay results with target locus genotype identifies ' genotypes of the target locus that alter the cellular phenotype.
  • the phenotypic characteristic can be responsiveness of the cell to 0 a xenobiotic, and the method can thus include the antecedent step of contacting the coisogenic cell collection with a xenobiotic.
  • the cells of the collection are coisogenic at a target selected from the group consisting of: CYP1A2, CYP2C17, CYP2D6, CYP2E, CYP3A4, CYP4A11, CYP1B1 , CYP1A1, CYP2A6, CYP2A13, CYP2B6, CYP2C8, CYP2C9, CYP11A, CYP2C19, CYP2F1, CYP2J2, CYP3A5, CYP3A7, 5 CYP4B1 , CYP4F2, CYP4F3, CYP6D1 , CYP6F1 , CYP7A1 , CYP7A1 , CY
  • the correlations can thereafter optionally be collected into at least one dataset, typically one or more relational databases, usefully recorded on a computer- readable medium.
  • the invention provides a method of predicting a phenotypic characteristic of a cell based upon its genotype at a target locus. The method comprises using the cell's genotype at the target locus, or a unique identifier thereof, as a query to retrieve from a dataset data that report a correlated phenotypic characteristic, wherein the dataset includes such correlations for at least five cells that are coisogenic at o the target locus; the retrieved phenotypic characteristic provides a prediction of the cell's phenotypic characteristic.
  • the term "cell” intends a eukaryotic cell. Unless otherwise made explicitly clear by context, the singular term “cell” equally intends a plurality of genetically identical cells, such as a plurality of cells from a clonal eukaryotic cell line.
  • a "cultured cell” is a eukaryotic cell (or clonal eukaryotic cell line) that is maintained alive in vitro in nutrient media, or that has previously been propagated in vitro in nutrient media for at least one doubling.
  • a "target locus” is a genomic region that includes all exons of an expressed protein.
  • two genotypically distinct cells are “coisogenic” with respect to one another if derived from a common ancestor cell and engineered to differ from one another in genomic sequence at a predetermined target locus.
  • the genomic sequence differences at the target locus must be sufficient to alter the amino acid sequence encoded at the target locus by at least one amino acid.
  • coisogenic permits of changes as between the genomes of the genotypically distinct cells additional to the changes at the target locus.
  • Exceptionally coisogenic cells are coisogenic cells that differ in genomic sequence by no more than 0.05%, excluding changes at the target locus.
  • Perfectly coisogenic cells are coisogenic cells that differ in genomic sequence by no more than 0.005%, excluding changes at the target locus.
  • Cells, or genetic alterations, therein are said to be "legacy-free” if lacking in heterologous genetic elements within 10 kilobases of an engineered genomic sequence alteration. When used with respect to coisogenic cells, the cells are legacy-free if lacking in heterologous genetic elements within 10 kilobases of any codon of the target locus.
  • heterologous genetic elements are sequences of greater than 25 consecutive nucleotides that derive from - and that can thus be shown to be present in - species different from that from which the coisogenic cells derive; heterologous genetic elements thus include, inter alia, all genetic elements derived from prokaryotic cells, including prokaryotic genomic DNA; genetic elements derived from prokaryotic episomes, including fertility factors; genetic elements derived from bacteriophage; as well as genetic elements from eukaryotic viruses.
  • the term “collection”, as applied to cells intends that the cells are in sufficient spatial proximity to one another as readily and contemporaneously to be subject to the same experimental protocol.
  • library is intended to be synonymous with “collection” in all respects.
  • xenobiotic intends a foreign compound introduced into a biological system, such as an inorganic or organic compound foreign to the cell or organism under study, or a compound naturally present in the cell or organism under study but administered by nonnatural routes or at unnatural concentrations.
  • the present invention is made possible by our recent discovery of methods and compositions, to be described in further detail below, for creating site-specific mutations in genomic DNA of eukaryotic cells, including mammalian cells, at efficiencies and with a precision not hitherto achievable using homologous recombination or earlier approaches based upon oligonucleotide-mediated gene repair.
  • the methods permit point mutations to be targeted with high efficiency to genomic DNA incubated in cellular extracts, such as artificial chromosomes incubated in cellular extracts, and also permit mutations to be targeted with high efficiency directly into the chromosomes of cultured cells.
  • the efficiency is sufficiently high as to obviate the concomitant insertion of selectable markers or other exogenous DNA, permitting cells with defined mutations to be created legacy-free.
  • the invention provides a collection of at least 5 genotypically distinct cells, typically as a collection of at least 5 genotypically distinct eukaryotic cell lines.
  • Each of the genotypically distinct cells (or cell lines) is coisogenic to the others of the genotypically distinct cells (or cell lines) in the collection at a common target locus.
  • each of the genotypically distinct cells can be separately assayed.
  • the cultured cells of the invention can be any eukaryotic cell amenable to in vitro culture.
  • human cells have particular utility, particularly for pharmacogenomic uses. Also very useful, particularly for structure-activity studies, are cells from related primates, such as chimpanzee, monkeys (including rhesus macaque), baboon, 5 orangutan, and gorilla, and those from rodents typically used as laboratory models, such as rats, mice, hamsters and guinea pigs. Cells can also usefully be from lagomorphs, such as rabbits; and from larger mammals, such as livestock, including horses, cattle, sheep, pigs, goats, and bison.
  • primates such as chimpanzee, monkeys (including rhesus macaque), baboon, 5 orangutan, and gorilla
  • rodents typically used as laboratory models such as rats, mice, hamsters and guinea pigs.
  • Cells can also usefully be from lagomorphs, such as rabbits; and from larger mammals, such as livestock, including horses, cattle, sheep, pigs,
  • Plant cells from fowl such as chickens, geese, ducks, turkeys, pheasant, ostrich and pigeon; fish such as zebrafish, salmon, tilapia, catfish, trout and bass; o and domestic pet species, such as dogs and cats.
  • Plant cells for which coisogenic cell collections can usefully be constructed according to the methods of the present invention include, for example, experimental model plants, such as Chlamydomonas reinhardtii, Physcomitrella patens, and Arahidopsis thaliana; crop plants such as cauliflower (Brassica oleracea), artichoke (Cynara scolymus); fruits such as apples (Malus, e.g.
  • melo melo
  • nuts such as walnut, Juglans, e.g. regia; peanut, Arachis hypogeae), orange (Citrus, e.g. maxima), peach (Prunus, e.g. Prunus persica), pear (Pyra, e.g. communis), plum (Prunus, e.g. domestica), strawberry (Fragaria, e.g. moschata or vesca), tomato (Lycopersicon, e.g. esculentum); leaves and forage, such as alfalfa (Medicago, e.g. sativa or truncatula), cabbage (e.g.
  • Brassica oleracea ), endive (Cichoreum, e.g. endivia), leek (Allium, e.g. porrum), lettuce (Lactuca, e.g. sativa), spinach (Spinacia, e.g. oleraceae), tobacco (Nicotiana, e.g. tabacum); roots, such as arrowroot (Maranta, e.g. arundinacea), beet (Beta, e.g. vulgaris), carrot (Daucus, e.g. carota), cassava (Manihot, e.g. esculenta), turnip (Brassica, e.g.
  • oilseeds such as beans (Phaseolus, e.g. vulgaris), pea (Pisum, e.g. sativum), soybean (Glycine, e.g. max), cowpea (Vigna unguiculata), mothbean (Vigna aconitifolia), wheat (Thticum, e.g. aestivum), sorghum (Sorghum e.g. bicolor), barley (Hordeum, e.g.
  • cell collections of the present invention can also usefully be drawn from lower eukaryotes, such as yeasts, particularly Saccharomyces cerevisiae, Schizosaccharomyces pombe, Pichia species, such as methanolica, Ustillago maydis, and Candida species, from roundworms, such as C. elegans, from zebra fish, and from Drosophila melanogaster.
  • yeasts particularly Saccharomyces cerevisiae, Schizosaccharomyces pombe
  • Pichia species such as methanolica, Ustillago maydis
  • Candida species from roundworms, such as C. elegans, from zebra fish, and from Drosophila melanogaster.
  • Eukaryotic cell lines from which coisogenic collections of the present invention may be created are readily available from a wide variety of sources known in the art, including the American Type Culture Collection (Manassas, VA, USA), the Deutsche Sammlung von Mikroorganismen und Zellkulturen (DSMZ, German Collection of Microorganisms and Cell Cultures), and the Riken Cell bank of Japan; 472 such culture collections are listed at http://wdcm.nig.ac.jp/hpcc.html.
  • Specialized cell collections are also well known, and include the NIGMS (National Institute of General Medical Studies) Human Genetic Cell Repository, the NIA Aging Cell Repository, the Autism Research Resource, the ADA Cell Repository Maturity Onset Diabetes Collection, and the HBDI Cell Repository Juvenile Diabetes Collection, all of which are maintained at the Coriell Institute for Medical Studies (Camden, NJ, USA).
  • Specialized yeast collections include the National Collection of Yeast Cultures (Institute of Food Research, Norwich Research Park, Colney, Norwich, UK).
  • genotypically distinct cells need not be immortalized, or otherwise capable of indefinite propagation.
  • the collection includes at least 5 coisogenic cells (typically, as clonal cell lines). Higher assay throughput is often obtained when the collection includes greater than 5, such as 6, 7, 8, 9, or 10 genotypically distinct, coisogenic cells. Collections of 24 coisogenic cells can conveniently be disposed in a 24 well culture plate; collections of 96 coisogenic cells can conveniently be arrayed in a 96 well microtiter dish. With recent development of microtiter dishes with footprint identical to that of the standard microtiter dish, but with higher well density, collections of 384, 864, 1536, 3456, 6144, and as many as 9600 coisogenic cells can readily and usefully be present in the cell collections of the present invention.
  • the collections need not necessarily contain such even numbers of genotypically distinct exceptionally coisogenic cells, and can thus include any number of genotypically distinct coisogenic cells greater than or equal to 5, including 6, 7, 8, 9, 10, 11 , 12, 13, 14, 15, 16, 17, 18, 19, 20, 30, 40, 50, 60, 70, 75, 80, 85, 90, 95, 100, 200, 300, 400, 500 or more.
  • At least five of the genotypically distinct cells of the collections of the present invention are coisogenic at a common, predetermined, target locus.
  • the target locus can be any protein-encoding locus of the cell.
  • preferred targets for pharmacogenomic studies encode proteins known to be involved in drug resistance and/or drug metabolism.
  • coisogenic cells have genomic sequence differences at the target locus that are sufficient to occasion change of at least one amino acid at the target locus.
  • the genotypically distinct cells of the collection are coisogenic to the others of the genotypically distinct cells of the collection.
  • the methods and compositions for creating the coisogenic cells which are further described below, readily permit the legacy-free substitution, addition, or deletion of as few as 1 and as many as 3 consecutive nucleotides in the genomic DNA of the target locus.
  • Alterations can include, for example, substitutions of one, two or three contiguous nucleotides, thus effecting a change in the amino acid encoded by one codon or by two adjacent codons. Since the standard genetic code is well known, the nucleotide changes required to effect change from any given codon to one that encodes any other 5 desired amino acid would be apparent to the skilled artisan; examples are also presented herein below.
  • one predetermined amino acid residue is commonly targeted for change in each of the coisogenic cells; with a minimum of 20 genotypically distinct cells in the collection, each of the commonly occurring natural amino 0 acids can be present in the collection at the target residue.
  • Residues that are particularly informative as targets are those that occur in the protein at locations of known structural and/or functional importance, such as within highly structured, ligand-binding domains.
  • the genotypically distinct cells can differ not at the identical residue, but at successive amino acids of the target protein.
  • each genotypically distinct cell can contain a single alanine substitution.
  • the first cell of the collection can have alanine substituted for residue 2; the second cell of the collection can have alanine substituted for residue 3; the third cell of the collection can have alanine substituted for residue 4, efc.
  • the coisogenic cells of the cell collection present an in vivo alanine scan of the o entire protein sequence, permitting ready identification of critical residues of the target protein.
  • Any amino acid can be used as the substitute in such an embodiment, with the choice dictated by the known chemical and biological properties of the naturally occurring amino acids.
  • praline can be substituted to effect disruption of 5 secondary structures, such as beta sheets or alpha helices
  • tyrosine can be substituted to provide substrates for tyrosine-kinase mediated post-translational modification
  • glutamic acid can be substituted to increase local charge density.
  • Alterations can also include introduction of a termination codon. Because any codon of the target locus can be targeted, coisogenic cells can be collected that each o individually possess a single engineered termination codon, but that collectively present consecutive, single amino acid truncations from the carboxy terminus of the target protein. Alterations can also include insertion of an amino acid, through targeted insertion of a novel codon between two existing codons.
  • Alterations can, in other embodiments, include frameshift mutations, caused by insertion or deletion of 1 or 2 nucleotides.
  • Frameshift can lead to truncation or 5 elongation, depending upon presence of termination codons in the new reading frame.
  • Introduction of compensating frameshifts e.g., insertion of a single nucleotide followed, at some distance downstream, by deletion of a single nucleotide, can lead to alteration of a series of amino acids between the mutated nucleotides.
  • the collection can include cells that are coisogenic at a first residue of the target locus, with the collection including all possible amino acids at that first target residue, with the collection further including cells that have substitutions at other residues of the target locus. o Greater differences can be achieved by targeting changes iteratively to the target locus using the methods of the present invention.
  • the coisogenic cells are legacy-free.
  • our methods for constructing coisogenic cell collections can alter genomic DNA without concomitant insertion of heterologous nucleic acids, such as selectable markers, prokaryotic genetic elements, o bacteriophage genetic elements, or eukaryotic viral elements, at the target locus. Because such heterologous nucleic acid close to the target locus can cause unpredictable changes in expression and/or activity of the target protein, they are disfavored, although permitted, in certain embodiments of the cell collections of the present invention.
  • heterologous nucleic acids such as selectable markers, prokaryotic genetic elements, o bacteriophage genetic elements, or eukaryotic viral elements
  • the coisogenic cells of the present invention will, on occasion, have accumulated genetic 5 differences at other than the target locus. Such differences are permissible.
  • the coisogenic cells of the collections of the present invention are "exceptionally coisogenic", differing in genomic sequence by no more than 0.05%, excluding changes at the target locus. In other embodiments, the cells are "perfectly coisogenic", differing in genomic sequence by no o more than about 0.005%, excluding changes at the target locus.
  • the exceptionally coisogenic cell collections and perfectly coisogenic cell collections of the present invention can each, additionally, be legacy-free.
  • the coisogenic cells of the cell collections of the present invention can also include intentional genetic changes at locations in the genome other than the target locus. 5 For example, mutations can be targeted to a second target locus, creating cell lines that are coisogenic at several targets.
  • markers can usefully, but optionally, be included, at a site other than the target locus.
  • Such marker can be common to all cells in the collection, for example by prior introduction into a cellular o ancestor common to all of the genotypically distinct cells, can be unique to each genotype, or can be common to some, but not to all, genotypically distinct cells in the collection.
  • a selectable marker can commonly be included in all of the genotypically distinct cells of the collection to prevent overgrowth, either by cells of the same lineage, or by other species.
  • Selectable markers are well known, and the choice 5 thereof will depend upon the species from which the genotypically distinct cells of the collection are derived.
  • Selectable markers for use in mammalian cells e.g., include markers that confer resistance to neomycin (G418), blasticidin, hygromycin or to zeocin; other well-known selections are based upon the purine salvage pathway.
  • Selectable markers in yeast include a variety of auxotrophic markers, such as alleles of URA3, HIS3, 0 LEU2, TRP1 and LYS2.
  • unique markers can be introduced into each of the genotypically distinct cells of the collection, allowing each genotypically distinct cell (typically, cell line) in the collection readily to be distinguished.
  • the sequence can encode substrate-independent 5 proteinaceous fluorophores with distinct emission spectra. See, e.g., Palm ef al., "Spectral Variants of Green Fluorescent Protein,” in Green Fluorescent Proteins, Conn (ed.), Methods Enzymol. vol. 302, pp. 378 - 394 (1999)), the disclosure of which is incorporated herein by reference.
  • the markers can also be intended to distinguish the cells at the nucleic o acid, rather than protein, level (genetic "bar codes"). If such bar codes are flanked by priming sites that are common to all of the bar codes of distinct sequence, a single amplification reaction (e.g., by PCR), can be used to stoichiometrically to amplify all bar codes, the presence and/or frequencies of which can thereafter readily be assayed. See, e.g., U.S. Patent No. 6,046,002. 5 Other genetic alterations that can usefully be made outside the target locus include those that facilitate assay of the cells of the coisogenic cell collection of the present invention, as will be discussed below.
  • the target locus for the coisogenic cell collections of the present invention can be any locus believed to contribute to a relevant cellular or organismic phenotype, and o thus usefully includes all proteins that are presently subject to drug screening assays (e.g.,
  • G protein coupled receptors protein kinases, zinc finger-containing transcription factors), or pharmacogenomic analysis (such as ApoE, presenilin 1 , presenilin 2, p53, efc).
  • Particularly useful targets in certain embodiments of the present invention are loci that encode proteins that affect drug responsiveness, in part because the clinical phenotype can readily be 5 correlated with a cellular phenotype, permitting ready assay in vitro.
  • the cell collections of the present invention can usefully be coisogenic at loci that encode any one of the P450 enzymes, which are known significantly to affect the metabolism of many, if not most, therapeutic agents.
  • the cytochrome P450 superfamily includes a large number (as many as 60 0 in human beings) of separate, but related, monooxygenases that play a central role in oxidative metabolism of a wide range of compounds, including therapeutic drugs. Although the number of known P450 enzymes is large, and the endogenous substrates of most unknown, a half dozen or so appear to be responsible for metabolism of the vast majority of prescribed and over-the-counter drugs: CYP1A2, CYP2C17, CYP2D6, CYP2E ("CYP2E1"), CYP3A4, and CYP4A11. For recent reviews, see Anzenbacher ef al., "Cytochromes P450 and metabolism of xenobiotics," Cell. Mol. Life Sci. 58(5-6):737-47 (2001), and Drug. Ther. Bull. 38(12):93-5 (2000).
  • the cell collections of the present invention can thus usefully be coisogenic at CYP1A2 (cytochrome P450, subfamily I (aromatic compound-inducible), polypeptide 2) (also known as CP12, P3-450, P450(PA)).
  • CYP1A2 cytochrome P450, subfamily I (aromatic compound-inducible), polypeptide 2)
  • This gene the human homologue of which is located about 25 kb away from CYP1A1 on chromosome 15 (at 15q22-qter), encodes a member of the cytochrome P450 superfamily of enzymes closely related to CYP1 A1.
  • the gene is aromatic compound-inducible, and is known to metabolize acetaminophen in human beings to the cytotoxic metabolite N-acetylbenzoquinoneimine (NABQI), Thatcher ef al., Cancer Gene Ther. 7(4):521-5 (2000).
  • NABQI N-acetylbenzoquinoneimine
  • CYP2C17 can also usefully be targeted.
  • CYP2D6 also known as CPD6, CYP2D, CYP2D@, P450C2D, P450-DB1 encodes cytochrome P450, subfamily IID (debrisoquine, sparteine, etc., -metabolizing), polypeptide 6, and is known to metabolize as many as 20% of commonly prescribed drugs; the cell collections of the present invention can usefully be coisogenic at this locus.
  • the enzyme's substrates include debrisoquine, an adrenergic-blocking drug; sparteine and propafenone, both anti-arrhythmic drugs; and amitryptiline, an anti-depressant.
  • the gene is highly polymorphic in the population; certain alleles result in the poor metabolizer phenotype, characterized by a decreased ability to metabolize the enzyme's substrates.
  • the gene is located near two cytochrome P450 pseudogenes on chromosome 22q13.1.
  • CYP2E (earlier denominated CPE1 , CYP2E1 , P450-J, P450C2E) encodes cytochrome P450, subfamily HE (ethanol-inducible), located in the human genome at 10q24.3-qter, and can usefully be targeted in constructing coisogenic cell collections of the present invention.
  • This P450 enzyme localizes to the endoplasmic reticulum and is induced by ethanol, the diabetic state, and starvation.
  • the enzyme metabolizes both endogenous substrates, such as ethanol, acetone, and acetal, as well as exogenous substrates including benzene, carbon tetrachloride, ethylene glycol, and nitrosamines which are premutagens found in cigarette smoke. Due to its many substrates, this enzyme may be involved in such varied processes as gluconeogenesis, hepatic cirrhosis, diabetes, and cancer.
  • CYP3A4 also known as CP34, NF-25, P450C3, P450PCN1
  • cytochrome P450, subfamily IIIA nifedipine oxidase
  • the enzyme encoded by CYP3A4 localizes to the endoplasmic reticulum and its expression is induced by glucocorticoids and some pharmacological agents. This enzyme is involved in the metabolism of approximately half the drugs used today, including 0 nifedipine, acetaminophen, codeine, cyclosporin A, diazepam and erythromycin. The enzyme also metabolizes some steroids and carcinogens.
  • Vinca alkaloids are important chemotherapeutic agents, and their pharmacokinetic properties display significant interindividual variations, possibly due to CYP3A4-mediated metabolism. See, Yao ef al., "Detoxication of vinca alkaloids by human 5 P450 CYP3A4-mediated metabolism: implications for the development of drug resistance," J. Pharmacol. Exp. Ther. 294(1 ):387-95 (2000).
  • CYP3A3A3 This gene is part of a cluster of cytochrome P450 genes on chromosome 7q21.1.
  • CYP3A3A3 another CYP3A gene, CYP3A3, was thought to exist; however, it is now thought that this sequence represents a transcript variant of CYP3A4.
  • o CYP4A11 also called CP4Y, CYP4A2, CYP4AII
  • CYP4A11 encodes a member of the cytochrome P450 superfamily of enzymes. This protein localizes to the endoplasmic reticulum and hydroxylates medium-chain fatty acids such as laurate and myristate. 5 Other cytochrome P450 enzymes can also usefully be targeted.
  • CYP1B1 (synonyms: CP1B, GLC3A), another target at which the cell collections of the present invention can usefully be coisogenic, encodes cytochrome P450, subfamily I (dioxin-inducible), polypeptide 1 (glaucoma 3, primary infantile), located in the human genome at 2p21.
  • the P450 monooxygenase encoded by this gene localizes to the o endoplasmic reticulum and metabolizes procarcinogens such as polycyclic aromatic hydrocarbons and 17beta-estradiol. Mutations in this gene have been associated with primary congenital glaucoma; therefore it is thought that the enzyme also metabolizes a signaling molecule involved in eye development, possibly a steroid.
  • CYP1 B1 has been shown to be increased in an anti-estrogen-resistant breast cell line, Brockdorff et ah, Int. J. Cancer 5 88(6):902-6 (2000), and has been generally implicated in tumor drug resistance, Rochat ef al., "Human CYP1 B1 and anticancer agent metabolism: mechanism for tumor-specific drug inactivation?", J. Pharmacol. Exp. Ther. 296(2):537-41 (2001); McFadyen ef al., "Cytochrome P450 CYP1 B1 protein expression: a novel mechanism of anticancer drug resistance," Biochem Pharmacol. 62(2):207-12 (2001).
  • CYP1A1 cytochrome P450, subfamily I (aromatic compound-inducible), polypeptidel) (also known as AHH, AHRR, CP11 , CYP1, P1-450, P450-C, P450DX), the human homologue of which is located at 15q22-24, can also usefully be targeted.
  • Expression and activity of CYP1 A are known to be induced by some polycyclic aromatic hydrocarbons (PAHs), some of which are found in cigarette smoke, and the enzyme is able 5 to metabolize some PAHs to carcinogenic intermediates; the gene has specifically been associated with lung cancer risk.
  • PAHs polycyclic aromatic hydrocarbons
  • CYP1 A activity has been shown to be increased in a breast cell line resistant to the antiestrogen compound IC1 1827801, Brockdorff ef a/., "Increased expression of cytochrome p450 1 A1 and 1 B1 genes in anti-estrogen-resistant human breast cancer cell o lines," Int. J. Cancer 88(6):902-6 (2000), and has been suggested as a marker for sensitivity to anti-cancer drugs, Peters ef al., "A mutation in exon 7 of the human cytochrome P- 4501A1 gene as marker for sensitivity to anti-cancer drugs?", Br. J. Cancer 75(9):1397 (1997).
  • CYP2A6 Another target for which cell collections of the present invention can 5 usefully be coisogenic is CYP2A6, the human homologue of which is found at 19q13.2, encoding cytochrome P450, subfamily IIA (phenobarbital-inducible), polypeptide 6 (also known as CPA6, CYP2A3).
  • CYP2A6 encodes a P450 enzyme that localizes to the endoplasmic reticulum; its expression is induced by phenobarbital. The enzyme is known to hydroxylate coumarin, and also metabolizes nicotine, aflatoxin B1 , nitrosamines, and some o pharmaceuticals.
  • CYP2A6 allelic variants of CYP2A6 are said to have a "poor metabolizer" phenotype, meaning they do not efficiently metabolize drugs that are substantially metabolized by CYP2A6, such as coumarin, nicotine, or fluoxetine (Prozac®).
  • CYP2A6 is part of a large cluster of cytochrome P450 genes from the CYP2A, CYP2B and 5 CYP2F subfamilies on chromosome 19q.
  • CYP2A6 is predominantly responsible for the metabolism of nicotine to cotinine, and many allelic variants have been described. See, Zabetian et ai, "Functional variants at CYP2A6: new genotyping methods, population genetics, and relevance to studies of tobacco dependence," Am. J. Med. Genet. 96(5):638-45 (2000). o Another cytochrome P450 enzyme that can usefully be targeted in the coisogenic cell collections of the present invention is CYP2A13 (also known as CPAD), the human homologue of which is located at 19q13.2.
  • CYP2A13 is phenobarbital-inducible, and is highly active in the metabolic activation of a major tobacco-specific carcinogen, 4- (methylnitrosamino)-l -(3-pyridyl)-1 -butanone, with a catalytic efficiency much greater than 5 that of other human cytochrome P450 isoforms.
  • a major tobacco-specific carcinogen 4- (methylnitrosamino)-l -(3-pyridyl)-1 -butanone
  • Catalytic efficiency much greater than 5 that of other human cytochrome P450 isoforms.
  • CYP2B6 (alternatively denominated CPB6, IIB1 , P450, and CYPIIB6), o encoding cytochrome P450, subfamily IIA (phenobarbital-inducible), polypeptide 6, is located at 19q13.2 in the human genome, and is a useful target locus for the coisogenic cell collections of the present invention.
  • This P450 enzyme localizes to the endoplasmic reticulum and its expression is induced by phenobarbital.
  • the enzyme is known to metabolize some xenobiotics, such as the anti-cancer drugs cyclophosphamide and 5 ifosphamide.
  • Transcript variants for this gene have been described; however, it has not been resolved whether these transcripts are in fact produced by this gene or by a closely related pseudogene, CYP2B7. Both the gene and the pseudogene are located in the middle of a CYP2A pseudogene found in a large cluster of cytochrome P450 genes from the CYP2A, CYP2B and CYP2F subfamilies on chromosome 19q. CYP2B6 is though to o mediate the N-demethylation of (R)- and (S)-ketamine in human liver.
  • CYP2C8 (same as CPC8, P450 MP-12/MP-20) encoding cytochrome P450, subfamily IIC (mephenytoin 4-hydroxylase), polypeptide 8, is also a useful target for the coisogenic eukaryotic cell collections of the present invention.
  • This protein localizes to the endoplasmic reticulum and its expression is induced by phenobarbital.
  • the enzyme is 5 known to metabolize many xenobiotics, including the anticonvulsive drug mephenytoin, benzo(a)pyrene, 7-ethyoxycoumarin, and the anti-cancer drug paclitaxel (Taxol®).
  • CYP2C8 also metabolizes cerivastatin, which is a high potency, third generation synthetic statin with proven lipid-lowering efficacy.
  • CYP2C9 cytochrome P450, subfamily IIC (mephenytoin 4-hydroxylase), 5 polypeptide 9
  • CYP2C9 cytochrome P450, subfamily IIC (mephenytoin 4-hydroxylase), 5 polypeptide 9
  • CYP2C9 and UGT1A6 genotypes modulate the protective effect of aspirin on colon adenoma risk
  • Cancer Res. 61(9):3566-9 2001.
  • CYP11A (same as P450SCC, cytochrome P450C11A1), also usefully targeted in the coisogenic cell collections of the present invention, encodes a member of the cytochrome P450 superfamily of enzymes. This protein localizes to the mitochondrial inner 5 membrane and catalyzes the conversion of cholesterol to pregnenolone, the first and rate-limiting step in the synthesis of the steroid hormones.
  • the human homologue is located at 15q23-q24.
  • CYP2C19 (same as CPCJ, CYP2C, P450C2C, P450IIC19, microsomal monooxygenase, xenobiotic monooxygenase, mephenytoin 4'-hydroxylase, o flavoprotein-linked monooxygenase), encodes cytochrome P450, subfamily IIC
  • polypeptide 19 This protein localizes to the endoplasmic reticulum and is known to metabolize many xenobiotics, including the anticonvulsive drug mephenytoin, omeprazole, diazepam, proguanil, and some barbiturates.
  • the enzyme is also responsible for the polymorphic (NAT2*) acetylation of hydrazine and aromatic amine drugs, such as isoniazid, hydralazine, and sulfasalazine. Polymorphism within this gene is 5 associated with variable ability to metabolize mephenytoin, known respectively as the poor metabolizer phenotype and extensive metabolizer phenotype.
  • the gene is located within a cluster of cytochrome P450 genes on chromosome 10q24, at 10q24.1-q24.3.
  • CYP4B1, CYP4F2 found to catalyze hydroxylation and dealkylation of an H(1)-antihistamine prodrug, ebastine, Hashizume et al., 5 "A novel cytochrome p450 enzyme responsible for the metabolism of ebastine in monkey small intestine," Drug Metab. Dispos.
  • CYP4F3, CYP6D1 , CYP6F1 (related to CYP6D1 and involved in pyrethroid detoxification in insects), CYP7A1 , CYP8, CYP11A, CYP11B1, CYP11B2 , CYP17, CYP19, CYP21A2, CYP24, CYP27A1 , and CYP51.
  • CYP4F3, CYP6D1 , CYP6F1 (related to CYP6D1 and involved in pyrethroid detoxification in insects), CYP7A1 , CYP8, CYP11A, CYP11B1, CYP11B2 , CYP17, CYP19, CYP21A2, CYP24, CYP27A1 , and CYP51.
  • ABC ATP-binding cassette
  • MRP multi-drug resistance phenotype
  • ABCB1 ATP-binding cassette, sub-family B (MDR/TAP), member 1
  • MDR1 multi drug resistance 0 1
  • P-GP P-glycoprotein
  • PGY1 PGY1
  • ABC20 GP170
  • the protein encoded by this gene is an ATP-dependent drug efflux pump for xenobiotic compounds with broad substrate specificity. It is responsible for decreased drug accumulation in multidrug-resistant cells and often mediates the development of resistance to anticancer drugs.
  • a number of studies have demonstrated a negative 5 correlation between Pgp expression levels and chemosensitivity or survival in a range of human malignancies. Lehne, "P-glycoprotein as a drug target in the treatment of multidrug resistant cancer," Curr. Drug Targets 1 (1 ):85-99 (2000).
  • P-glycoprotein is also expressed in normal tissues with excretory function such as liver, kidney and intestine. Apical expression of P-glycoprotein in such tissues 0 results in reduced drug absorption from the gastrointestinal tract and enhanced drug elimination into bile and urine. Moreover, expression of P-glycoprotein in the endothelial cells of the blood-brain barrier prevents entry of certain drugs into the central nervous system. Human P-glycoprotein has been shown to transport a wide range of structurally unrelated drugs such as digoxin, quinidine, cyclosporin and HIV-1 protease inhibitors. 5 Studies in humans indicate a particular importance of intestinal P-glycoprotein for bioavailability of the immunosuppressant cyclosporin.
  • Allelic variants of ABCB1 are known to affect its selectivity and/or activity. Hoffmeyer ef a/., "Functional polymorphisms of the human multidrug-resistance gene: multiple sequence variations and correlation of one allele with P-glycoprotein o expression and activity in vivo," Proc. Natl. Acad. Sci USA 97(7):3473-8 (2000); Choi ef al., "An altered pattern of cross-resistance in multidrug-resistant human cells results from spontaneous mutations in the mdrl (P-glycoprotein) gene," Ce// 53(4):519-29 (1988).
  • ABCB4 (ATP-binding cassette, sub-family B (MDR/TAP), member 4)(also known as MDR3, PGY3, ABC21 , MDR2/3, PFIC-3) (human homologue maps to 7q21.1), is 5 another useful target locus for the coisogenic cell collections of the present invention.
  • the membrane-associated protein encoded by this gene is a member of the superfamily of ATP-binding cassette (ABC) transporters.
  • ABCB4 is a member of the MDR/TAP subfamily.
  • Members of the MDR/TAP subfamily are involved in multidrug resistance as well as antigen presentation.
  • This gene encodes a full transporter and o member of the p-glycoprotein family of membrane proteins with phosphatidylcholine as its substrate.
  • ABCC1 -- ATP-binding cassette, sub-family C (CFTR/MRP), member 1 - is a member of the MRP subfamily of ATP- binding cassette (ABC) proteins, and is involved in multi-drug resistance.
  • This protein 5 functions as a multispecific organic anion transporter, with oxidized glutathione, cysteinyl leukotrienes, and activated aflatoxin B1 as known substrates.
  • This protein also transports glucuronides and sulfate conjugates of steroid hormones and bile salts.
  • Alternative splicing by exon deletion results in several splice variants but maintains the original open reading frame in all forms.
  • ABCC2 (same as DJS, MRP2, cMRP, ABC30, CMOAT, Canalicular multispecific organic anion transporter) encodes ATP-binding cassette, sub-family C (CFTR/MRP), member 2, and is a useful target locus for the coisogenic cell collections of the present invention.
  • ABCC2 is a member of the MRP subfamily of ATP binding cassette proteins, and is involved in multi-drug resistance. This protein is expressed in the 5 canalicular (apical) part of the hepatocyte and functions in biliary transport.
  • Known substrates include anticancer drugs such as vinblastine.
  • ABCC3 also known as MLP2, MRP3, ABC31 , CMOAT2, MOAT-D, EST90757
  • the o protein may play a role in the transport of biliary and intestinal excretion of organic anions.
  • splicing of this gene results in three known transcript variants.
  • a useful target for the coisogenic cell collections of the present invention is ATP-binding cassette, sub-family C (CFTR/MRP), member 4, ABCC4, also known as MRP4, MOATB, MOAT-B, EST170205.
  • the protein encoded by this gene is a member of the MRP subfamily of ABC transporters, and is involved in multi-drug resistance. 5
  • the protein may play a role in cellular detoxification as a pump for its substrate, organic anions.
  • ABCC4 MRP4
  • ABCC5 MRP5
  • thiopurine anticancer drugs such as o 6-mercatopurine and thioguanine
  • 9-(2-phosphonylmethoxyethyl)adenine this protein may be involved in resistance to thiopurines in acute lymphoblastic leukemia and antiretroviral nucleoside analogs in HIV-infected patients); ABCC6 (MRP6), MRP7 (CFTR), ABCC8 (MRP8), ABCC9, ABCC10, ABCC11 (same as HI, SUR, MRP8, PHHI, SUR1 , ABC36, HRINS), and ABCC12 (same as 5 MRP9).
  • EPHX1 epoxide hydrolase 1 , microsomal xenobiotic
  • EPHX2 epoxide hydrolase 2
  • LTA4H leukotriene A4 hydrolase
  • TRAG3 Texol® resistance associated gene 3, which is overexpressed in most melanoma cells and confers resistance to paclitaxel, Taxol®
  • GUSB beta-glucuronidase
  • TMPT thiopurine o methyltransferase
  • BCRP (breast cancer resistance protein, an ATP transporter), dihydropyrihidine dehydrogenase, HERG (involved in drug transport through potassium ion channels), hKCNE2 (involved in drug transport through potassium ion channels), UDP glucuronosyl transferase (UGT) (a hepatic metabolizing enzyme, a detoxifying enzyme for most carcinogens after different cytochrome P450 (CYP) isoform
  • Another protein usefully targeted in the coisogenic cell collections of the o present invention is the BCR-ABL fusion responsible for chronic myeloid leukemia.
  • the tyrosine kinase domain of the fusion protein is targeted by imatinib (Gleevec); allelic variants have been identified that confer polyclonal resistance to the drug.
  • beta tubulin Another protein usefully targeted in the coisogenic cell collections of the present invention is beta tubulin.
  • Paclitaxel is a tubulin-disrupting agent that binds 5 preferentially to beta-tubulin. Allelic variants of beta tubulin have been identified that confer resistance to paclitaxel. Giannakakou ef al., J. Biol. Chem. 272:17118-17125 (1997), incorporated herein by reference in its entirety.
  • the coisogenic cell collections of the present invention can usefully include l o cells that have, at the coisogenic target locus, the sequence of a naturally-occurring allele; this permits the phenotype conferred by the allele to be assessed without the confounding presence of other genetic differences at the target locus or elsewhere in the cellular genome. Accordingly, the coisogenic cell collections of the present invention can usefully include cells that have the naturally occurring (allelic) variants set forth in the following 15 tables.
  • angiotensin I working draft 1306 aa converting chromo17 enzyme J04144 (m)
  • CYP2D6*1 see CYP2D6*36
  • CYP2D6 CYP2D6*1 CYP2D6.1 1023CT; CYP2D6Z T107I; Deer Deer Masimirem
  • cytochrome X55765 (exonl and 503 aa
  • hypoaldosteronism GTG > GCG Val386Ala Congenital OMIM 124080 hypoaldosteronism CGG > TGG Arg181Tr Congenital OMIM 124080 p. hypoaldosteronism
  • cytochrome 508 aa cytochrome 508 aa
  • CTX2 polypeptide 1
  • CYP27 362 in the adrenodoxin binding region.

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Abstract

L'invention concerne des collections de cellules eucaryotes cultivées, notamment des cellules humaines, dans lesquelles les cellules sont co-isogéniques à un emplacement cible commun. Notamment, cette invention a trait à des collections de cellules co-isogéniques qui diffèrent au niveau de la séquence génomique de 0,05 % au maximum, ce qui exclut des changements au niveau de l'emplacement cible, à des collections dans lesquelles les cellules co-isogéniques diffèrent au niveau de la séquence génomique de 0,005 % au maximum, ce qui exclut des modifications au niveau de l'emplacement cible, et à des collections dans lesquelles les cellules manquent d'éléments génétiques hétérologues au sein de 10 kilobases de l'emplacement cible co-isogénique. Cette invention concerne également des kits renfermant les collections cellulaires, des méthodes et des kits d'élaboration desdites collections, et des méthodes d'utilisation de ces collections destinées à faciliter des analyses pharmacogénomiques. Les emplacements cibles préférés au niveau desquels les cellules sont co-isogéniques, contiennent des gènes qui modifient la résistance aux médicaments, la sensibilité aux médicaments, et/ou le métabolisme des médicaments.
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